A group of metabolic disorders known as lysosomal storage diseases (LSD) includes over forty genetic disorders, many of which involve genetic defects in various lysosomal hydrolases. Representative lysosomal storage diseases and the associated defective enzymes are listed in Table 1.
TABLE 1Lysosomal storage diseaseDefective enzymeAspartylglucosaminuriaAspartylglucosaminidaseFabryα-Galactosidase AInfantile Batten Disease* (CNL1)Palmitoyl Protein ThioesteraseClassic Late Infantile BattenTripeptidyl PeptidaseDisease* (CNL2)Juvenile Batten Disease* (CNL3)Lysosomal Transmembrane ProteinBatten, other forms* (CNL4-CNL8)Multiple gene productsCystinosisCysteine transporterFarberAcid ceramidaseFucosidosisAcid α-L-fucosidaseGalactosidosialidosisProtective protein/cathepsin AGaucher types 1, 2*, and 3*Acid β-glucosidase, orglucocerebrosidaseGM1 gangliosidosis*Acid β-galactosidaseHunter*Iduronate-2-sulfataseHurler-Scheie*α-L-IduronidaseKrabbe*Galactocerebrosidaseα-Mannosidosis*Acid α-mannosidaseβ-Mannosidosis*Acid β-mannosidaseMaroteaux-LamyArylsulfatase BMetachromatic leukodystrophy*Arylsulfatase AMorquio AN-Acetylgalactosamine-6-sulfatesulfataseMorquio BAcid β-galactosidaseMucolipidosis II/III*N-Acetylglucosamine-1-phosphotransferaseNiemann-Pick A*, BAcid sphingomyelinaseNiemann-Pick C*NPC-1Pompe*Acid α-glucosidaseSandhoff*β-Hexosaminidase BSanfilippo A*Heparan N-sulfataseSanfilippo B*α-N-AcetylglucosaminidaseSanfilippo C*Acetyl-CoA:α-glucosaminideN-acetyltransferaseSanfilippo D*N-Acetylglucosamine-6-sulfatesulfataseSchindler Disease*α-N-AcetylgalactosaminidaseSchindler-Kanzakiα-N-AcetylgalactosaminidaseSialidosisα-NeuramidaseSly*β-GlucuronidaseTay-Sachs*β-Hexosaminidase AWolman*Acid Lipase*CNS involvement
The hallmark feature of LSD is the abnormal accumulation of metabolites in the lysosomes which leads to the formation of large numbers of distended lysosomes in the perikaryon. A major challenge to treating LSD (as opposed to treating a liver-specific enzymopathy) is the need to reverse lysosomal storage pathology in multiple separate tissues. Some LSDs can be effectively treated by intravenous infusion of the missing enzyme, known as enzyme replacement therapy (ERT). For example, Gaucher type 1 patients have only visceral disease and respond favorably to ERT with recombinant glucocerebrosidase (Cerezyme®, Genzyme Corp.). However, patients with metabolic disease that affects the CNS (e.g., type 2 or 3 Gaucher disease) do not respond to intravenous ERT because the replacement enzyme is prevented from entering the brain by the blood brain barrier (BBB). Furthermore, attempts to introduce a replacement enzyme into the brain by direct injection have been unsuccessful in part due to enzyme cytotoxicity at high local concentrations (unpublished observations) and limited parenchymal diffusion rates in the brain (Pardridge, Peptide Drug Delivery to the Brain, Raven Press, 1991).
Alzheimer's disease (AD) is a disorder affecting the central nervous system (CNS) characterized by the accumulation of amyloid β-peptide (Aβ) due to decreased Aβ catabolism. As Aβ accumulates, it aggregates into extracellular plaques, causing impairment of synaptic function and loss of neurons. The pathology leads to dementia, loss of coordination, and death.
Gene therapy is an emerging treatment modality for disorders affecting the CNS, including LSDs and Alzheimer's disease. In this approach, restoration of the normal metabolic pathway and reversal of pathology occurs by transducing affected cells with a vector carrying a healthy version or a modified version of the gene.
CNS gene therapy has been facilitated by the development of viral vectors capable of effectively infecting post-mitotic neurons. For a review of viral vectors for gene delivery to the CNS, see Davidson et al. (2003) Nature Rev., 4:353-364. Adeno-associated virus (AAV) vectors are considered optimal for CNS gene therapy because they have a favorable toxicity and immunogenicity profile, are able to transduce neuronal cells, and are able to mediate long-term expression in the CNS (Kaplitt et al. (1994) Nat. Genet., 8:148-154; Bartlett et al. (1998) Hum. Gene Ther., 9:1181-1186; and Passini et al. (2002) J. Neurosci., 22:6437-6446).
A therapeutic transgene product, e.g., an enzyme, can be secreted by transduced cells and subsequently taken up by other cells, in which it then alleviates pathology. This process is known as cross-correction (Neufeld et al. (1970) Science, 169:141-146). However, the correction of pathology, such as storage pathology in the context of LSD, is typically confined to the immediate vicinity of the injection site because of limited parenchymal diffusion of the injected vector and the secreted transgene product (Taylor et al. (1997) Nat. Med., 3:771-774; Skorupa et al. (1999) Exp. Neurol., 160:17-27). Thus, neuropathology affecting multiple brain regions requires widespread vector delivery, using multiple spatially distributed injections, especially in a large brain such as human. This significantly increases the risk of brain damage. In addition, some regions of the brain may be difficult to access surgically. Thus, other modes of vector transport within the CNS, besides diffusion, would be beneficial.
When administered at axonal endings, some viruses are internalized and transported retrogradely along the axon to the nucleus. Neurons in one brain region are interconnected by axons to distal brain regions thereby providing a transport system for vector delivery. Studies with adenovirus, HSV, and pseudo-rabies virus have utilized trafficking properties of these viruses to deliver genes to distal structures within the brain (Soudas et al. (2001) FASEB J., 15:2283-2285; Breakefield et al. (1991) New Biol., 3:203-218; and deFalco et al. (2001) Science, 291:2608-2613).
Several groups have reported that the transduction of the brain by AAV serotype 2 (AAV2) is limited to the intracranial injection site (Kaplitt et al. (1994) Nat. Genet., 8:148-154; Passini et al. (2002) J. Neurosci., 22:6437-6446; and Chamberlin et al. (1998) Brain Res., 793:169-175). One recent report suggests that retrograde axonal transport of AAV2 can also occur in select circuits of the normal rat brain (Kaspar et al. (2002) Mol. Ther., 5:50-56). However, it is not known what specific parameters were responsible for the observed axonal transport, and whether sufficient and effective axonal transport would occur in a diseased neuron that is in a state of cellular dysfunction. Indeed, lesions observed in LSD neurons have been reported to interfere with or even block axonal transport (reviewed in Walkley (1998) Brain Pathol., 8:175-193), suggesting that disease-compromised neurons would not support trafficking of AAV along their axons.
Therefore, there is a need in the art to develop new therapeutic methods for treating metabolic disorders that affect the CNS.